Human embryonic stem cells (HuES) have the potential to generate an unlimited source of beta/islet cells for transplantation, however the biologic factors required for this process have been difficult to ascertain partly due to the lack of stage specific in vitro cell assays. Recently, we have developed a murine in vitro pancreatic stem/progenitor colony assay that we are using to identify factors required for beta cell /islet differentiation and expansion. Based on this murine assay, we propose in this study to develop a HuES based assay that would accelerate the discovery of previously unidentified but required human factors for the generation of beta cell/islets. The phase I goals are: 1) to translate our well-defined murine pancreatic progenitor colony assay to a human assay based on HuES cells, and 2) to create a reporter HuES cell line with insertion of enhanced green fluorescent protein (EGFP) that is driven by promoter of neurogenin (Ngn) 3, a marker for pancreatic endocrine progenitors. Once a HuES based progenitor assay is in place, we will then transition into phase II study to screen for proteins or molecules that could be used to expand the number of progenitors. Our ultimate intention is to commercialize the identified biological mediators for the purpose of cell replacement therapy or stimulation of regeneration of endogenous pancreatic [unreadable] cell progenitors to treat type 1 diabetes. Lay Abstract: Human embryonic stem cells (HuES) have the potential to generate an unlimited source of beta/islet cells for transplantation, however the biologic factors required for this process have been difficult to ascertain partly due to the lack of stage specific in vitro cell assays. This phase I proposal will establish an HuES cell based pancreatic progenitor assay for the purpose of screening for biological mediators in the future phase II study. Our ultimate intention is to commercialize the identified biological mediators for the purpose of cell replacement therapy or stimulation of regeneration of endogenous pancreatic [unreadable] cell progenitors to treat type 1 diabetes. [unreadable] [unreadable] [unreadable]